46th annual meeting of the Radiation Research Society Conference, Louisville, KY Optical imaging of living cells and intact tissues by non-linear, simultaneous multiphoton molecular excitation provides inherent three-dimensional diffraction-limited resolution while minimizing photodamage and peripheral photobleaching. The multiphoton laser scanning microscope raster-scans a focused, high repetition-rate femtosecond pulse train of infrared laser light across biological samples and efficiently detects the resulting multiphoton excited fluorescence. This development has recently allowed quantitative visualization of cellular processes and tissue architecture that had been problematic or impossible with conventional microscopies. The physical principles, aspects of instrumentation developments, and photophysical characterization of molecular fluorophores that have enabled multiphoton microscopy will be discussed. Examples such as neurotransmitter secretion, skin morphology, drug metabolism in the intact colon, and simultaneous metabolic and photosensitizer imaging in t umor spheroids demonstrate a range of applications pursued with this novel form of microscopy at the Developmental Resource for Biophysical Imaging and Opto-electronics (DRBIO) at Cornell. Supported at DRBIO by the NIH RR04224. M.G.N. is an NIH Postdoctoral Fellow supported by NRSA 1 F32 CA 72225-02.